Non-invasive methods of detecting target molecules

ABSTRACT

Embodiments of the present invention relate to non-invasive methods and compositions for collecting detecting, measuring, and identifying target molecules. In some embodiments, methods and compositions relate to target molecules in gastrointestinal lavage fluid or feces.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/344,399, filed Mar. 12, 2014; which is a national stage ofInternational Patent Application No. PCT/US2011/051269, filed Sep. 12,2011; the contents of each of the aforementioned applications areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

Embodiments of the present invention relate to non-invasive methods andcompositions for collecting, detecting, measuring, and identifyingtarget molecules. In some embodiments, methods and compositions relateto target molecules in gastrointestinal lavage fluid (GLF) or feces.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a compliant ANSItext file, which is approximately 231 KB in size. The information in theelectronic format of the Sequence Listing is incorporated herein byreference in its entirety.

BACKGROUND

Disorders associated with the gastrointestinal (GI) and hepatobiliarytracts and the organs/tissues associated with the GI tract includecancers such as gastric cancer, esophageal cancer, liver cancer, andpancreatic cancer. Pancreatic cancer (e.g., pancreatic adenocarcinoma),in particular, is a malignant growth of the pancreas that mainly occursin the cells of the pancreatic ducts. This disease is the ninth mostcommon form of cancer, yet it is the fourth and fifth leading cause ofcancer deaths in men and women, respectively. Cancer of the pancreas isalmost always fatal, with a five-year survival rate that is less than3%.

The most common symptoms of pancreatic cancer include jaundice,abdominal pain, and weight loss, which, together with other presentingfactors, are often nonspecific in nature. Thus, diagnosing pancreaticcancer at an early stage of tumor growth is often difficult and requiresextensive diagnostic work-up, often times incidentally discovered duringexploratory surgery. Endoscopic ultrasonography is an examplenon-surgical technique available for diagnosis of pancreatic cancer.However, reliable detection of small tumors, as well as differentiationof pancreatic cancer from focal pancreatitis, is difficult. The vastmajority of patients with pancreatic cancer are presently diagnosed at alate stage when the tumor has already extended beyond the pancreas toinvade surrounding organs and/or has metastasized extensively. Gold etal., Crit. Rev. Oncology/Hematology, 39:147-54 (2001), incorporatedherein by reference in its entirety. Late detection of the disease iscommon with the majority of patients being diagnosed with advanceddisease that often results in death; only a minority of patients aredetected with early stage disease.

Invasive techniques to diagnose disorders and diseases related to the GItract are inconvenient and expose a subject to significant risk.Accordingly, there is a need for non-invasive methods and compositionsfor the detection and identification of target molecules from the GItract or associated organs/tissues. In some embodiments, the targetmolecules may be evaluated to determine whether they are useful asbiomarkers associated with a particular characteristic, such as disease,predisposition to disease, positive response to a treatment regimen, orno response or negative response to a treatment regimen. In addition,biomarkers from the GI tract or associated organs/tissues may be used todetermine whether an individual has any of the particularcharacteristics listed above.

SUMMARY

Embodiments of the present invention relate to non-invasive methods andcompositions for collecting, detecting, measuring, and identifyingtarget molecules. In some embodiments, methods and compositions relateto target molecules in gastrointestinal lavage fluid (GLF) or feces.

Some embodiments include a method for assessing the physiological stateof a subject comprising: obtaining a gastrointestinal lavage fluid fromthe subject; and detecting a target molecule which originated fromoutside the gastrointestinal system in the gastrointestinal lavagefluid.

Some embodiments include a method for assessing the physiological stateof a subject comprising: obtaining a fecal sample from the subject; anddetecting a target molecule which originated from outside thegastrointestinal system in the fecal sample.

In some embodiments, the gastrointestinal lavage fluid is obtained fromthe subject by partially purging the subject's gastrointestinal system.

In some embodiments, the gastrointestinal lavage fluid comprises fecalmatter. In some embodiments, the fecal sample comprises agastrointestinal lavage fluid.

In some embodiments, the target molecule comprises a polypeptide,antibody, bile acid, metabolite, or glycan. In some embodiments, thetarget molecule comprises a biomarker. In some embodiments, thebiomarker is associated with a disease, a positive response totreatment, a partial response to treatment, a negative response totreatment, or no response to treatment. In some embodiments, the targetmolecule is associated with presence of a cancer or predisposition to acancer. In some embodiments, the cancer is pancreatic cancer, colorectalcancer, liver cancer, or gastric cancer. In some embodiments, the targetmolecule originated from an accessory digestive gland. In someembodiments, the accessory digestive gland is salivary glands, pancreas,gallbladder, or liver.

Some embodiments include administering a lavage fluid to the subject. Insome embodiments, lavage fluid is administered orally. In someembodiments, the lavage fluid comprises an ingredient selected from thegroup consisting of polyethylene glycol, magnesium sulfate, sodiumsulfate, potassium sulfate, magnesium citrate, ascorbic acid, sodiumpicosulfate, and bisacodyl. In some embodiments, the lavage fluid isselected from the group consisting of GOLYTELY, HALFLYTELY, NULYTELY,SUPREP, FLEET'S PHOSPHO-SODA, magnesium citrate, and their genericequivalents.

Some embodiments include performing a colonoscopy on the subject.

In some embodiments, the subject is mammalian. In some embodiments, thesubject is human.

Some embodiments include a method for identifying a biomarkercomprising: obtaining a test gastrointestinal lavage fluid from aplurality of test subjects having a condition or physiological state ofinterest and a control gastrointestinal lavage fluid from a plurality ofcontrol subjects who do not have said condition or physiological state;determining the level of at least 5 target molecules in the testgastrointestinal lavage fluid and the control gastrointestinal lavagefluid, and identifying a target molecule which is present atsignificantly different levels in the test gastrointestinal lavage fluidrelative to the levels in the control gastrointestinal lavage fluid,thereby identifying a biomarker.

In some embodiments, the gastrointestinal lavage fluid comprises fecalmatter.

In some embodiments, the target molecules are selected form the groupconsisting of polypeptides, bile acids, antibodies, metabolites,glycans, and a combination thereof.

Some embodiments include determining the level of at least 10 targetmolecules in the test gastrointestinal lavage fluid and the controlgastrointestinal lavage fluid. Some embodiments include determining thelevel of at least 20 target molecules in the test gastrointestinallavage fluid and the control gastrointestinal lavage fluid. Someembodiments include determining the level of at least 30 targetmolecules in the test gastrointestinal lavage fluid and the controlgastrointestinal lavage fluid. Some embodiments include determining thelevel of at least 50 target molecules in the test gastrointestinallavage fluid and the control gastrointestinal lavage fluid. Someembodiments include determining the level of at least 100 targetmolecules in the test gastrointestinal lavage fluid and the controlgastrointestinal lavage fluid.

In some embodiments, the biomarker is associated with a disease, apositive response to treatment, a partial response to treatment, anegative response to treatment or no response to treatment.

In some embodiments, the biomarker is associated with the presence of acancer or predisposition to a cancer. In some embodiments, the cancer ispancreatic cancer, liver cancer, or gastric cancer.

In some embodiments, at least one target molecule originated from anaccessory digestive gland. In some embodiments, the accessory digestivegland is salivary glands, pancreas, gallbladder, or liver.

Some embodiments include administering a lavage fluid to the testsubjects and the control subjects. In some embodiments, the lavage fluidis administered orally. In some embodiments, the lavage fluid comprisesan ingredient selected from the group consisting of polyethylene glycol,magnesium sulfate, sodium sulfate, potassium sulfate, magnesium citrate,ascorbic acid, sodium picosulfate, and bisacodyl. In some embodiments,the lavage fluid is selected from the group consisting of GOLYTELY,HALFLYTELY, NULYTELY, SUPREP, and FLEET'S PHOSPHO-SODA, magnesiumcitrate, and their generic equivalents.

Some embodiments include performing a colonoscopy on the test subjectsand control subjects.

In some embodiments, the test subjects and control subjects aremammalian In some embodiments, the test subjects and control subjectsare human.

Some embodiments include a method for identifying a biomarkercomprising: obtaining a test fecal sample from a plurality of testsubjects having a condition of interest and a control fecal sample froma plurality of control subjects and; determining the level of at least 5target molecules in the test fecal sample and the control fecal sample,identifying a target molecule which is present at significantlydifferent levels in the test fecal sample relative to the levels in thecontrol fecal sample, thereby identifying a biomarker.

In some embodiments, the fecal sample comprises a gastrointestinallavage fluid.

In some embodiments, the target molecules are selected from the groupconsisting of polypeptides, bile acids, antibodies, metabolites,glycans, and a combination thereof.

Some embodiments include determining the level of at least 10 targetmolecules in the test fecal sample and the control fecal sample. Someembodiments include determining the level of at least 20 targetmolecules in the fecal sample and the control fecal sample. Someembodiments include determining the level of at least 30 targetmolecules in the fecal sample and the control fecal sample. Someembodiments include determining the level of at least 50 targetmolecules in the fecal sample and the control fecal sample. Someembodiments include determining the level of at least 100 targetmolecules in the fecal sample and the control fecal sample.

In some embodiments, the biomarker is associated with a disease, apositive response to treatment, or a negative response to treatment.

In some embodiments, the biomarker is associated with the presence of acancer or predisposition to a cancer. In some embodiments, the cancer ispancreatic cancer, colorectal cancer, liver cancer, or gastric cancer.

In some embodiments, at least one target molecule originated from anaccessory digestive gland. In some embodiments, the accessory digestivegland is salivary glands, pancreas, gallbladder, or liver.

In some embodiments, the test subjects and control subjects aremammalian In some embodiments, the test subjects and control subjectsare human.

Some embodiments include a kit for detecting a target molecule in agastrointestinal lavage fluid comprising: a lavage fluid for oraladministration to a subject; a vessel for collecting thegastrointestinal lavage fluid from the subject; and an agent fordetecting a target molecule which originated from outside thegastrointestinal system.

Some embodiments include a kit for detecting a target molecule in afecal sample comprising: a lavage fluid for oral administration to asubject; a vessel for collecting the fecal sample from the subject; andan agent for detecting a target molecule which originated from outsidethe gastrointestinal system.

Some embodiments include a protease inhibitor.

In some embodiments, the target molecule comprises a polypeptide,antibody, bile acid, metabolite, or glycan. In some embodiments, thetarget molecule comprises a biomarker. In some embodiments, thebiomarker is associated with a disease, a positive response totreatment, or a negative response to treatment.

In some embodiments, the target molecule is associated with presence ofa cancer or predisposition to a cancer. In some embodiments, the canceris pancreatic cancer, liver cancer, colorectal cancer, or gastriccancer.

In some embodiments, the target molecule originated from an accessorydigestive gland. In some embodiments, the accessory digestive gland issalivary glands, pancreas, gallbladder, or liver.

In some embodiments, the lavage fluid comprises an ingredient selectedfrom the group consisting of polyethylene glycol, magnesium sulfate,sodium sulfate, potassium sulfate, magnesium citrate, ascorbic acid,sodium picosulfate, and bisacodyl. In some embodiments, the lavage fluidis selected from the group consisting of GOLYTELY, HALFLYTELY, NULYTELY,SUPREP, FLEET'S PHOSPHO-SODA, magnesium citrate, and their genericequivalents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a graph of the relative abundance of various glycoproteinderived glycan structures present in a fraction of a gastrointestinallavage fluid. Derivatized glycans were eluted from a C18 reverse phasecolumn on a Q-TOF MS at about 20-25% acetonitrile in 0.2% formic acid.The mass spectrometer was scanned in MS-only mode from m/z 150-2000every second to acquire the derivatized glycan profile data.

FIG. 2 depicts a graph of the relative abundance of compounds, includingmetabolites such as cholic acid, present in a fraction of agastrointestinal lavage fluid. Data were acquired on a Waters Q-TOF massspectrometer using input from an LC system, and using MassLynx software.The MS scanned over the mass range from m/z 100 m/z to 2000 everysecond.

DETAILED DESCRIPTION

Embodiments of the present invention relate to non-invasive methods andcompositions for collecting, detecting, measuring, and identifyingtarget molecules. In some embodiments, methods and compositions relateto target molecules in gastrointestinal lavage fluid (GLF) or feces.

Gastrointestinal lavage is widely used as a lower gastrointestinal (GI)tract preparation for colonoscopy or colorectal surgery (see e.g.,DiPalma J A. et al., (1984) Gastroenterology 86:856-60), incorporatedherein by reference in its entirety). Particular pathophysiologies ofintestinal diseases have been investigated by measuring proteins in GLF(Evgenikos N, et al. (2000) Br J Surg 87:808-13; Brydon W G, Ferguson A.(1992) Lancet 340: 1381-2; Choudari C P, et al. (1993) Gastroenterology104: 1064-71; Ferguson A, et al. (1996) Gut 38:120-4; Handy L M, et al.(1996) Scand J Gastroenterol 31:700-5; and Stanley A J, et al. (1996)Gastroenterology 111:1679-82), each incorporated herein by reference inits entirety).

Measurements of fecal proteins can be useful for investigating variouspathophysiologies such as protein-losing enteropathy and mucosalinflammation. However, while feces may be used in some embodimentsdescribed herein, GLF is preferred over feces as a sample for detectingand identifying biomarkers because GLF contains smaller amounts ofsubstances that interfere with assays, and destruction of protein bydigestive enzymes and bacterial proteases is less in GLF than a fecalsample because of its quick transit through the GI tract. In addition,it is possible to estimate the rate of protein release from the mucosa,because the rate of fluid passage along the gut can be estimated.

In some embodiments, a GLF can be produced by orally administering alavage fluid to a subject that causes a large volume of fluid to passthrough the intestinal tract, the lavage fluid can contain a mixture ofsalts and other materials such as polyethylene glycol and bisacodyl. Thelavage fluid causes an influx of liquid into the colon that causes aflushing out of solids. Lavage fluids are commonly used to causeclearing of the GI tract as is commonly used in preparation for acolonoscopy and other methods used to examine the GI tract. Theseliquids that are flushed out or remain in the largely cleared GI tractare useful to evaluate a variety of diseases due to the continuity ofthe mouth to the anus along the GI tract. Consequently, any and allorgans, including the GI tract, which deposit fluids into the GI tractare candidates for the methods and compositions provided herein.

GI Tract and Associated Organs/Tissues

Some of the methods and compositions provided herein relate to the GItract and organs/tissues associated with the GI tract includingaccessory digestive glands. As is well known in the art, the GI tractincludes the upper GI tract and lower GI tract. The upper GI tractincludes the oral or buccal cavity, esophagus, stomach and duodenum. Thelower GI tract includes the jejunum, ileum and the large intestine andthe anus. The large intestine includes the appendix, cecum, colon, andrectum.

Organs and tissues associated with the GI tract include structuresoutside the GI tract. Examples of such structures include accessorydigestive organs such as salivary glands, e.g., parotid salivary glands,submandibular salivary glands, and sublingual salivary glands, pancreas,e.g., exocrine pancreas, gallbladder, bile duct, and liver. Moreexamples of structures associated with the GI tract and outside the GItract include the pancreatic duct, biliary tree, and bile duct.

Gastrointestinal Lavage Fluid (GLF)

Generally, a lavage fluid can be orally administered to a subject, theoral lavage fluid passes through the GI tract of the subject, and theresulting GLF is collected from the subject. As used herein, the term“subject” can include an animal, such as a mammal, such as a human Asnoted above, GLF provides a cleaner sampling of the GI tract than theexamination of feces/stool samples. GLFs appear to mitigate variabilityrelated to food intake, type and digestive status.

Some embodiments described herein include analysis of a GLF fordetecting a target molecule or for screening, triage, disease detection,diagnosis, prognosis, response to treatment, selection of treatment andpersonalized medicine for diseases and pathological conditions of thegastrointestinal tract or associated organs/tissues. Some embodimentsinclude analysis of a GLF sample for eliminating particular diseases andpathological conditions from the possible diseases or conditions fromwhich a subject may be suffering. Some embodiments include analysis of aGLF for indicating the need for further testing for diagnosis. Moreembodiments include the analysis of GLF to establish a new diseasediagnosis, further classifying a previous diagnosis, determining thesensitivity to potential treatment regimens, and/or evaluating theresponse to previous or ongoing treatment regimens.

Methods for Obtaining a GLF

Some embodiments of the methods and compositions provided herein includeobtaining a GLF from a subject. Methods of obtaining a GLF are wellknown in the art. For example, during medical and or diagnosticprocedures such as sigmoidoscopy, colonoscopy, radiographic examination,preparation for patients undergoing bowel surgery, it is important thatthe bowels and colon be thoroughly purged and cleaned. In particular, itis essential that as much fecal matter as possible be removed from thecolon to permit adequate visualization of the intestinal mucosa. This isimportant prior to, for example, diagnostic procedures such as flexiblesigmoidoscopy or colonoscopy, diagnostic examinations widely performedto screen patients for diseases of the colon. In addition, it isimportant that the intestines be cleansed thoroughly in order to obtainsatisfactory radiographs of the colon. The same condition also applieswhen the colon is preoperatively prepared for surgery, where removal offecal waste materials is critically important for patient safety. Toprepare the colon for endoscopic exam, current cleaning proceduresinclude orthograde colonic lavage. Orthograde lavage can include orallyadministering a lavage composition to a subject comprising 4 L of apolyethylene glycol/electrolyte solution (U.S. Patent ApplicationPublication No. 20070298008, incorporated by reference in its entirety).Some embodiments include antegrade lavage and retrograde lavage.

Generally, oral lavage compositions include solutions of electrolytes,such as sodium, potassium and magnesium salts of sulfate, bicarbonate,chloride, phosphate or citrate. Some such compositions may also includepolyethylene glycol, which can act as a non-absorbable osmotic agent.Generic compositions include polyethylene glycol with an electrolytesolution, optionally also including bisacodyl, or ascorbic acid, andcompositions including sulfate salts such as sodium sulfate, magnesiumsulfate, or potassium sulfate. In some embodiments, an oral lavage fluidcan include magnesium citrate. In some embodiments, an oral lavage fluidcan include sodium picosulfate. One example composition of an orallavage solution comprising polyethylene glycol with an electrolytesolution is GOLYTELY (Braintree Labs. Inc.). GOLYTELY is formulatedaccording to the following: polyethylene glycol 59 g, sodium sulfate5.68 g, sodium bicarbonate 1.69 g, sodium chloride 1.46 g, potassiumchloride 0.745 g and water to make up one liter (Davis et al. (1980)Gastroenterology 78:991-995, incorporated by reference in its entirety).Ingestion of GOLYTELY produces a voluminous, liquid stool with minimalchanges in the subject's water and electrolyte balance. Another exampleof an oral lavage composition comprising polyethylene glycol with anelectrolyte solution is NULYTELY (Braintree Labs. Inc.). An example orallavage composition comprising polyethylene glycol with an electrolytesolution and bisacodyl is HALFLYTELY (Braintree Labs. Inc.). An exampleoral lavage composition comprising sulfate salts, such as sodiumsulfate, magnesium sulfate, or potassium sulfate is SUPREP (BraintreeLabs. Inc.). An example composition of an oral lavage solutioncomprising polyethylene glycol with an electrolyte solution and ascorbicacid is MOVIPREP (Salix Pharmaceuticals, Inc.).

Polyethylene glycol is effective as an oral lavage composition whenlarge amounts of polyethylene glycol are administered in large volumesof a dilute salt solution. Usually about 250-400 g polyethylene glycolare administered to the subject in about 4 L of an electrolyte solutionin water. Oral administration of polyethylene glycol can be used toproduce a bowel movement over a period of time, e.g., overnight. Thedose required will vary, but from about 10-100 g of polyethylene glycolin 8 oz. of water can be effective. A dose of from about 68-85 g ofpolyethylene glycol can be effective to produce an overnight bowelmovement, without profuse diarrhea. A volume of a solution ofpolyethylene glycol in an isotonic fluid can be an effective amount ofan osmotic laxative. Volumes from about 0.5 L to about 4 L can beeffective. Preferably the effective volume is between about 1.5 L andabout 2.5 L. Oral administration of 2 L of isotonic solution iseffective.

More examples of oral lavage compositions include hypertonic solutionsof non-phosphate salts with an osmotic laxative agent such aspolyethylene glycol (U.S. Pat. App. No. 20090258090, incorporated byreference in its entirety). Mixtures of sulfate salts that omitphosphates, for example, effective amounts of one or more of thefollowing sulfate salts Na₂SO₄, MgSO₄, and K₂SO₄ can be effective (e.g.,SUPREP). Some embodiments include about 0.1 g to about 20.0 g Na₂SO₄,and from about 1.0 g to 10.0 g Na₂SO₄ may be useful. Dosage amounts ofMgSO₄ from about 0.01 g to about 40.0 g can be effective. Doses of fromabout 0.1 g to about 20.0 g Na₂SO₄ may also be advantageously used, aswell as dosages of 1.0 to 10.0 g. Dosage amounts of K₂SO₄ from about0.01 g to about 20.0 g can be effective to produce purgation, and dosesof from about 0.1 g to about 10.0 g and from about 0.5 g to about 5.0 gK₂SO₄ may also be useful. Addition of an osmotic laxative agent, such aspolyethylene glycol (PEG) may improve the effectiveness of the abovesalt mixtures. Doses of PEG from about 1.0 g to about 100 g PEG areeffective. Doses from about 10.0 g to about 50 g of PEG are alsoeffective, as is a dose of about 34 g. For ease of administration, theabove mixture of salts can be dissolved in a convenient volume of water.A volume of less than one liter of water can be well tolerated by mostsubjects. The mixture can be dissolved in any small volume of water, andvolumes of between 100 and 500 ml are useful. The effective dose may bedivided and administered to the patient in two or more administrationsover an appropriate time period. Generally, administration of two dosesof equal portions of the effective dose, separated by 6 to 24 hoursproduces satisfactory purgation. Some embodiments include cessation ofnormal oral intake during a defined period before and duringadministration of an oral lavage composition.

Some lavage compositions include a laxative, such as bisacodyl. In someembodiments, a laxative can be co-administered to a subject with alavage composition. As will be understood, such co-administration caninclude, for example, administration of a laxative up to several hoursbefore administration of a lavage composition to a subject,administration of a laxative with the administration of a lavagecomposition to a subject, or administration of a laxative up to severalhours after administration of a lavage composition to a subject.Examples of laxatives and their effective doses include Aloe, 250-1000mg.; Bisacodyl, about 5-80 mg.; Casanthranol, 30-360 mg.; Cascaraaromatic fluid extract, 2-24 ml.; Cascara sagrada bark, 300-4000 mg.;Cascada sagrada extract, 300-2000 mg.; Cascara sagrada fliuid extract,0.5-5.0 ml.; Castor oil, 15-240 ml.; Danthron, 75-300 mg.; DehydrocholicAcid, 250-2000 mg; Phenolphthalein, 30-1000 mg.; Sennosides A and B,12-200 mg.; and Picosulfate, 1-100 mg.

More examples of lavage compositions include aqueous solutions ofconcentrated phosphate salts. The aqueous phosphate salt concentrateproduces an osmotic effect on the intra-luminal contents of the GItract, evacuation of the bowel occurs with a large influx of water andelectrolytes into the colon from the body. One exemplary compositioncomprises 480 g/L monobasic sodium phosphate and 180 g/L dibasic sodiumphosphate in stabilized buffered aqueous solution (FLEET'S PHOSPHO-SODA,C. S. Fleet Co., Inc.). Subjects are typically required to take 2-3 ozdoses of this composition, separated by a three to 12 hour interval fora total of 6 ounces (180 ml).

GLF may be collected from a subject before, during, or after a medicalor diagnostic procedure. In some embodiments, a subject may collect GLF,for example, using a receptacle such as a toilet insert which capturesthe fluid. Enzyme inhibitors and denaturants may be used to preserve thequality of the GLF. In some embodiments, the pH of the sample may beadjusted to help stabilize the samples. In some embodiments, GLF samplesmay be further treated to remove some or all solids and/or bacteria,such as by centrifugation. In some embodiments, the GI tract may not befully purged by administration of an oral lavage composition. Forexample, a portion of a complete dose of an oral lavage compositionrequired to fully purge the GI tract of a subject can be administered tothe subject. In some embodiments, a GLF can comprise fecal matter. Inmore embodiments, fecal matter can comprise a GLF.

Target Molecules

Some embodiments described herein relate to methods of detecting targetmolecules in samples obtained from the GI tract or compositions usefulfor such detection. As used herein, “target molecule” includes anymolecule that can be detected or measured or identified in a sample fromthe GI tract. Such samples include a GLF from a subject, and a fecalsample from a subject. Examples of target molecules include moleculessuch as peptides, polypeptides, proteins, mutant proteins, proteinsgenerated from alternative splicing, modified proteins, such aspost-translationally modified proteins e.g., glycosylated proteins,phosphorylated proteins, antibodies (e.g., autoantibodies, IgG, IgA, andIgM), antibody fragments, sugars, e.g., monosaccharides, disaccharides,oligosaccharides, and glycans, lipids, small molecules, e.g.metabolites, pharmaceutical compositions, metabolized pharmaceuticalcompositions, and pro-drugs. More examples of target molecules includebile salts and bile acids, e.g., cholic acid. More examples includechenodeoxycholic acid, glycocholic acid, taurocholic acid, deoxycholicacid, and lithocholic acid. Target molecules can originate in the GItract and outside the GI tract, e.g., from organs and/or tissuesassociated with the GI tract, such as accessory digestive glands. Insome embodiments, cells including their fragments and their otherbiproducts, e.g., red blood cells, white blood cells, and endothelialcells, organisms, e.g., bacteria, protozoans, and viruses and viralparticles can be detected in a GLF or fecal samples. In someembodiments, the target molecules may be any of the proteins or portionsthereof listed in any of Tables 1-10 herein or a portion thereof. Insome embodiments, the portion of the proteins listed in any of Tables1-10 can comprise at least 10, at least 15, at least 20, at least 25, atleast 50, or more than 50 consecutive amino acids of any the proteinslisted in Tables 1-10. In some embodiments, the target molecules maycomprise, consist essentially of, or consist of a polypeptide of one ofSEQ ID NO.s: 01-804. A polypeptide consisting essentially of one of SEQID NO.s: 01-804 may include additional amino acids or substituentsbeyond those in SEQ ID NO.s: 01-804 where such additional amino acids orsubstituents do not prevent the polypeptide from being detectable.

Target molecules also include biomarkers. As used herein, the term“biomarker” includes any target molecule present in a GLF or fecalsample that is associated with a disease, predisposition to disease,positive response to a particular treatment regimen, no response to aparticular treatment regimen, or negative response to a particulartreatment regimen. In some embodiments, a biomarker can be identified,measured and/or correlated with a diagnosis or prognosis of a disease.

In some embodiments, a target molecule is a component of a fluid of thesubject selected from the group consisting of blood, saliva, gastricjuice, hepatic secretion, bile, duodenal juice, and pancreatic juice. Insome embodiments, a target molecule is expressed in the uppergastrointestinal tract of the subject, or the lower gastrointestinaltract of the subject. In some embodiments, a target molecule isexpressed at a location in the subject selected from the groupconsisting of buccal cavity, esophagus, stomach, biliary tree,gallbladder, duodenum, jejunum, ileum, appendix, cecum, colon, rectum,and anal canal.

In some embodiments, a target molecule does not include a protein orother compound found in a GLF, for example, lactoferrin,eosinophil-derived neurotoxin, eosinophil cationic protein, bilirubin(Bil), alkaline phosphatase (ALP), aspartate aminotransferase,hemoglobin, or eosinophil peroxidase. In some embodiments, a targetmolecule does not include a protein found in feces, for example,heptaglobulin, hemopexin, a-2-macroglobulin, cadherin-17, calprotectin,carcinoembryogenic antigen, metalloproteinase-1 (TIMP-1), S100A12,K-ras, or p53. In some embodiments, a target molecule does not include aprotein found in pancreatic juice, for example, anterior gradient-2(AGR2), insulin-like growth factor binding protein-2, CEACAM6, MUC1,CA19-9, serine proteinase-2 (PRSS2) preproprotein, pancreaticlipase-related protein-1 (PLRP1), chymotrypsinogen B (CTRB), elastase 3B(ELA3B), tumor rejection antigen (pg96), azurocidin,hepatocarcinoma-intestine-pancreas/pancreatitis-associated-protein I(HIP/PAP-I), matrix metalloproteinase-9 (MMP-9), oncogene DJ1 (DJ-1), oralpha-1B-glycoprotein precursor (A1BG).

Methods for Characterizing Target Molecules

Some embodiments of the methods and compositions provided herein includecharacterizing a target molecule in a GLF or fecal sample.Characterizing a target molecule can include, for example, identifying atarget molecule, detecting a target molecule, and/or quantifying atarget molecule. Methods to identify, detect and quantify targetmolecule are well known in the art.

Some embodiments include identifying, determining the presence orabsence of a target molecule, and/or quantifying a target molecule,wherein the target molecule comprises a peptide, polypeptide, and/orprotein. Such target molecules may be characterized by a variety ofmethods such as immunoassays, including radioimmunoassays, enzyme-linkedimmunoassays and two-antibody sandwich assays as described herein. Avariety of immunoassay formats, including competitive andnon-competitive immunoassay formats, antigen capture assays andtwo-antibody sandwich assays also are useful (Self and Cook, (1996)Curr. Opin. Biotechnol. 7:60-65, incorporated by reference in itsentirety). Some embodiments include one or more antigen capture assays.In an antigen capture assay, antibody is bound to a solid phase, andsample is added such that antigen, e.g., a target molecule in GLF or afecal sample, is bound by the antibody. After unbound proteins areremoved by washing, the amount of bound antigen can be quantitated, ifdesired, using, for example, a radioassay (Harlow and Lane, (1988)Antibodies A Laboratory Manual Cold Spring Harbor Laboratory: New York,incorporated by reference in its entirety) Immunoassays can be performedunder conditions of antibody excess, or as antigen competitions, toquantitate the amount of antigen and, thus, determine a level of atarget molecule in GLF or a fecal sample.

Enzyme-linked immunosorbent assays (ELISAs) can be useful in certainembodiments provided herein. An enzyme such as horseradish peroxidase(HRP), alkaline phosphatase (AP), β-galactosidase or urease can belinked, for example, to an anti-HMGB1 antibody or to a secondaryantibody for use in a method of the invention. A horseradish-peroxidasedetection system can be used, for example, with the chromogenicsubstrate tetramethylbenzidine (TMB), which yields a soluble product inthe presence of hydrogen peroxide that is detectable at 450 nm. Otherconvenient enzyme-linked systems include, for example, the alkalinephosphatase detection system, which can be used with the chromogenicsubstrate p-nitrophenyl phosphate to yield a soluble product readilydetectable at 405 nm. Similarly, a β-galactosidase detection system canbe used with the chromogenic substrateo-nitrophenyl-β-D-galactopyranoside (ONPG) to yield a soluble productdetectable at 410 nm, or a urease detection system can be used with asubstrate such as urea-bromocresol purple (Sigma Immunochemicals).Useful enzyme-linked primary and secondary antibodies can be obtainedfrom a number of commercial sources such as Jackson Immuno-Research(West Grove, Pa.) as described further herein.

In certain embodiments, a target molecule in GLF or a fecal sample canbe detected and/or measured using chemiluminescent detection. Forexample in certain embodiments, specific antibodies to a particulartarget molecule are used to capture the target molecule present in thebiological sample, e.g., GLF or a fecal sample and an antibody specificfor the target molecule-specific antibodies and labeled with anchemiluminescent label is used to detect the target molecule present inthe sample. Any chemiluminescent label and detection system can be usedin the present methods. Chemiluminescent secondary antibodies can beobtained commercially from various sources such as Amersham. Methods ofdetecting chemiluminescent secondary antibodies are known in the art.

Fluorescent detection also can be useful for detecting a target moleculein certain methods provided herein. Useful fluorochromes include, DAPI,fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin,R-phycoerythrin, rhodamine, Texas red and lissamine Fluorescein orrhodamine labeled antibodies, or fluorescein- or rhodamine-labeledsecondary antibodies can be useful in the invention.

Radioimmunoassays (RIAs) also can be useful in certain methods providedherein. Such assays are well known in the art. Radioimmunoassays can beperformed, for example, with ¹²⁵I-labeled primary or secondary antibody(Harlow and Lane, supra, 1988).

A signal from a detectable reagent can be analyzed, for example, using aspectrophotometer to detect color from a chromogenic substrate; aradiation counter to detect radiation, such as a gamma counter fordetection of ¹²⁵I; or a fluorometer to detect fluorescence in thepresence of light of a certain wavelength. Where an enzyme-linked assayis used, quantitative analysis of the amount of a target molecule can beperformed using a spectrophotometer such as an EMAX Microplate Reader(Molecular Devices; Menlo Park, Calif.) in accordance with themanufacturer's instructions. The assays of the invention can beautomated or performed robotically, if desired, and that the signal frommultiple samples can be detected simultaneously.

In some embodiments, capillary electrophoresis based immunoassays(CEIA), which can be automated if desired, may be used to detect and/ormeasure the target molecule. Immunoassays also can be used inconjunction with laser-induced fluorescence as described, for example,in Schmalzing and Nashabeh, Electrophoresis 18:2184-93 (1997), and Bao,J. Chromatogr. B. Biomed. Sci. 699:463-80 (1997), each incorporated byreference in its entirety. Liposome immunoassays, such as flow-injectionliposome immunoassays and liposome immunosensors, also can be used todetect target molecules or to determine a level of a target moleculeaccording to certain methods provided herein (Rongen et al., (1997) J.Immunol. Methods 204:105-133, incorporated by reference in itsentirety).

Sandwich enzyme immunoassays also can be useful in certain embodiments.In a two-antibody sandwich assay, a first antibody is bound to a solidsupport, and the antigen is allowed to bind to the first antibody. Theamount of a target molecule is quantitated by measuring the amount of asecond antibody that binds to it.

Quantitative Western blotting also can be used to detect a targetmolecule or to determine a level of target molecule in a method providedherein. Western blots can be quantitated by well known methods such asscanning densitometry. As an example, protein samples areelectrophoresed on 10% SDS-PAGE Laemmli gels. Primary murine monoclonalantibodies, for example, against a target molecule are reacted with theblot, and antibody binding confirmed to be linear using a preliminaryslot blot experiment. Goat anti-mouse horseradish peroxidase-coupledantibodies (BioRad) are used as the secondary antibody, and signaldetection performed using chemiluminescence, for example, with theRenaissance chemiluminescence kit (New England Nuclear; Boston, Mass.)according to the manufacturer's instructions. Autoradiographs of theblots are analyzed using a scanning densitometer (Molecular Dynamics;Sunnyvale, Calif.) and normalized to a positive control. Values arereported, for example, as a ratio between the actual value to thepositive control (densitometric index). Such methods are well known inthe art as described, for example, in Parra et al., J. Vasc. Surg.28:669-675 (1998), incorporated herein by reference in its entirety.

As described herein, immunoassays including, for example, enzyme-linkedimmunosorbent assays, radioimmunoassays and quantitative westernanalysis, can be useful in some embodiments for detecting a targetmolecule or determining a level of a target molecule. Such assaystypically rely on one or more antibodies. As would be understood by theskilled artisan, methods described herein can be used to readilydistinguish proteins with alternative forms of post-translationmodifications, e.g., phosphorylated proteins, and glycosylated proteins.

Target molecules, such as protein target molecules, can be characterizedby a variety of methods. Proteins, polypeptides and peptides can beisolated by a variety of methods well known in the art, such as proteinprecipitation, chromatography (e.g., reverse phase chromatography, sizeexclusion chromatography, ion exchange chromatography, liquidchromatography), affinity capture, and differential extractions.

Isolated proteins can under go enzymatic digestion or chemical cleavageto yield polypeptide fragments and peptides. Such fragments can beidentified and quantified. A particularly useful method for analysis ofpolypeptide/peptide fragments and other target molecules is massspectrometry (U.S. Pat. App. No. 20100279382, incorporated by referencein its entirety). A number of mass spectrometry-based quantitativeproteomics methods have been developed that identify the proteinscontained in each sample and determine the relative abundance of eachidentified protein across samples (Flory et al., Trends Biotechnol.20:S23-29 (2002); Aebersold, J. Am. Soc. Mass Spectrom. 14:685-695(2003); Aebersold, J. Infect. Dis. 187 Suppl 2:S315-320 (2003);Patterson and Aebersold, Nat. Genet. 33 Suppl, 311-323 (2003); Aebersoldand Mann, Nature 422:198-207 (2003); Aebersold, R. and Cravatt, TrendsBiotechnol. 20:S1-2 (2002); Aebersold and Goodlett, Chem. Rev. 101,269-295 (2001); Tao and Aebersold, Curr. Opin. Biotechnol. 14:110-118(2003), each incorporated by reference in its entirety). Generally, theproteins in each sample are labeled to acquire an isotopic signaturethat identifies their sample of origin and provides the basis foraccurate mass spectrometric quantification. Samples with differentisotopic signatures are then combined and analyzed, typically bymultidimensional chromatography tandem mass spectrometry. The resultingcollision induced dissociation (CID) spectra are then assigned topeptide sequences and the relative abundance of each detected protein ineach sample is calculated based on the relative signal intensities forthe differentially isotopically labeled peptides of identical sequence.

More techniques for identifying and quantifying target moleculeslabel-free quantitative proteomics methods. Such methods include: (i)sample preparation including protein extraction, reduction, alkylation,and digestion; (ii) sample separation by liquid chromatography (LC orLC/LC) and analysis by MS/MS; (iii) data analysis includingpeptide/protein identification, quantification, and statisticalanalysis. Each sample can be separately prepared, then subjected toindividual LC-MS/MS or LC/LC-MS/MS runs (Zhu W. et al., J. ofBiomedicine and Biotech. (2010) Article ID 840518, 6 pages, incorporatedby reference in its entirety). An example technique includes LC-MS inwhich the mass of a peptide coupled with its correspondingchromatographic elution time as peptide properties that uniquely definea peptide sequence, a method termed the accurate mass and time (AMT) tagapproach. Using LC coupled with Fourier transform ion cyclotronresonance (LC-FTICR) MS to obtain the chromatographic and high massaccuracy information, peptide sequences can be identified by matchingthe AMT tags to previously acquired LC-MS/MS sequence information storedin a database. By taking advantage of the observed linear correlationbetween peak area of measured peptides and their abundance, thesepeptides can be relatively quantified by the signal intensity ratio oftheir corresponding peaks compared between MS runs (Tang, K., et al.,(2004) J. Am. Soc. Mass Spectrom. 15:1416-1423; and Chelius, D. andBondarenko, P. V. (2002) J. Proteome Res. 1: 317-323, incorporated byreference in their entireties). Statistics tools such as the Student'st-test can be used to analyse data from multiple LC-MS runs for eachsample (Wiener, M. C., et al., (2004) Anal. Chem. 76:6085-6096,incorporated by reference in its entirety). At each point of acquisitiontime and m/z, the amplitudes of signal intensities from multiple LC-MSruns can be compared between two samples to detect peptides withstatistically significant differences in abundance between samples.

As will be understood, a variety of mass spectrometry systems can beemployed in the methods for identifying and/or quantifying apolypeptide/peptide fragments. Mass analyzers with high mass accuracy,high sensitivity and high resolution include, ion trap, triplequadrupole, and time-of-flight, quadrupole time-of-flight massspectrometeres and Fourier transform ion cyclotron mass analyzers(FT-ICR-MS). Mass spectrometers are typically equipped withmatrix-assisted laser desorption (MALDI) or electrospray ionization(ESI) ion sources, although other methods of peptide ionization can alsobe used. In ion trap MS, analytes are ionized by ESI or MALDI and thenput into an ion trap. Trapped ions can then be separately analyzed by MSupon selective release from the ion trap. Fragments can also begenerated in the ion trap and analyzed. Sample molecules such asreleased polypeptide/peptide fragments can be analyzed, for example, bysingle stage mass spectrometry with a MALDI-TOF or ESI-TOF system.Methods of mass spectrometry analysis are well known to those skilled inthe art (see, e.g., Yates, J. (1998) Mass Spect. 33:1-19; Kinter andSherman, (2000) Protein Sequencing and Identification Using Tandem Mass.Spectrometry, John Wiley & Sons, New York; and Aebersold and Goodlett,(2001) Chem. Rev. 101:269-295, each incorporated by reference in itsentirety).

For high resolution polypeptide fragment separation, liquidchromatography ESI-MS/MS or automated LC-MS/MS, which utilizes capillaryreverse phase chromatography as the separation method, can be used(Yates et al., Methods Mol. Biol. 112:553-569 (1999), incorporated byreference in its entirety). Data dependent collision-induceddissociation (CID) with dynamic exclusion can also be used as the massspectrometric method (Goodlett, et al., Anal. Chem. 72:1112-1118 (2000),incorporated by reference in its entirety).

Once a peptide is analyzed by MS/MS, the resulting CID spectrum can becompared to databases for the determination of the identity of theisolated peptide. Methods for protein identification using singlepeptides have been described previously (Aebersold and Goodlett, Chem.Rev. 101:269-295 (2001); Yates, J. Mass Spec. 33:1-19 (1998), David N.et al., Electrophoresis, 20 3551-67 (1999), each incorporated byreference in its entirety). In particular, it is possible that one or afew peptide fragments can be used to identify a parent polypeptide fromwhich the fragments were derived if the peptides provide a uniquesignature for the parent polypeptide. Moreover, identification of asingle peptide, alone or in combination with knowledge of a site ofglycosylation, can be used to identify a parent glycopolypeptide fromwhich the glycopeptide fragments were derived. As will be understood,methods that include MS can be used to characterize proteins, fragmentsthereof, as well as other types of target molecules described herein.

Some embodiments can include enriching proteins and/or protein fractionsof a GLF. Example methods can include protein precipitation,chromatography, such as reverse phase chromatography, size exclusionchromatography, ion exchange chromatography, liquid chromatography, aswell as affinity capture, differential extraction methods andcentrifugation. Proteins and/or protein fractions can be furtherexamined using intact protein methods such as top-down proteomics or gelchromatography such as SDS-PAGE.

Some embodiments include identifying, determining the presence orabsence of a target molecule, and/or quantifying a target molecule,wherein the target molecule comprises a glycosylated protein and/orglycan. Glycosylated proteins and glycans can be analyzed by variousmethods well known in the art. Changes in glycosylation can beindicative of a disease or disease state. Thus, particular targetmolecules can include particular glycosylated proteins and/or glycans.As will be understood, a glycan may be a component of a glycoprotein,proteoglycan or other glycan containing compounds.

Some embodiments include identifying, determining the presence orabsence of a target molecule, and/or quantifying a target molecule,wherein the target molecule comprises a metabolite. Metabolites may beanalyzed in a GLF or fecal sample using a variety of methods. Forexample, a GLF or fecal sample can be analyzed for metabolites usingmethods such as chromatography. Some components of the metabolomeinclude bile acids and other small organic compounds. Metabolites caninclude peptides that are present in a GLF or fecal sample.

Methods for Identifying Biomarkers

In some embodiments, the target molecules detected in GLF or a fecalsample can be evaluated to determine whether they are biomarkersassociated with a particular condition, such as a disease, orphysiological state. Such biomarkers can be indicative for a particulardisease, predisposition to disease, prognosis, positive response to aparticular treatment regimen, or negative response to a particulartreatment regimen. In some embodiments, the presence or absence, orlevel of a biomarker can be associated with a particular condition, suchas a disease, or physiological state. In some embodiments, the presenceor absence, or level of a biomarker can be statistically correlated tothe particular condition, such as a disease, or physiological state. Insome embodiments, a physiological state can include a disease. In someembodiments, a biomarker can be correlated to a particular condition,such as disease, or physiological state by comparing the level ofexpression of a biomarker in a subject having a condition, such as adisease, or physiological state with the level of expression of thebiomarker in a subject not having a condition or physiological state.

In some embodiments, the differential expression of a biomarker in asubject having a condition compared to the expression of a biomarker ina subject not having a condition is indicative of a condition orphysiological state. As used herein, “differential expression” refers toa difference in the level of expression of a biomarker in a subjecthaving a condition, such as a disease, or physiological state and asubject not having the condition, such as a disease, or physiologicalstate. For example, the term “differential expression” can refer to thepresence or absence of a biomarker in a subject having a condition, suchas a disease, or physiological state compared with a subject not havinga condition or physiological state. In some embodiments, differentialexpression can refer to a difference in the level of expression of abiomarker in a subject having a condition, such as disease, orphysiological state compared with the level of expression of a biomarkerin a subject not having the condition, such as a disease, orphysiological state.

Differences in the level of a biomarker can be determined by measuringthe amount or level of expression of a biomarker using methods providedherein. In some embodiments, differential expression can be determinedas the ratio of the levels of one or more biomarker products betweenreference subjects/populations having or not having a condition orphysiological state, wherein the ratio is statistically significant.Differential expression between populations can be determined to bestatistically significant as a function of p-value. When using p-valueto determine statistical significance, a biomarker, the p-value ispreferably less than 0.2. In another embodiment, the biomarker isidentified as being differentially expressed when the p-value is lessthan 0.15, 0.1, 0.05, 0.01, 0.005, 0.0001 etc. When determiningdifferential expression on the basis of the ratio, a biomarker productis differentially expressed if the ratio of the level of expression in afirst sample as compared with a second sample is greater than or lessthan 1.0. For example, a ratio of greater than 1.0 for example includesa ratio of greater than 1.1, 1.2, 1.5, 1.7, 2, 3, 4, 10, 20 and thelike. A ratio of less than 1.0, for example, includes a ratio of lessthan 0.9, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05 and the like. In anotherembodiment, a biomarker can be differentially expressed if the ratio ofthe mean of the level of expression of a first population as comparedwith the mean level of expression of the second population is greaterthan or less than 1.0. For example, a ratio of greater than 1.0 includesa ratio of greater than 1.1, 1.2, 1.5, 1.7, 2, 3, 4, 10, 20 and the likeand a ratio less than 1.0, for example includes a ration of less than0.9, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05 and the like. In another embodiment abiomarker is differentially expressed if the ratio of its level ofexpression in a first sample as compared with the mean of the secondpopulation is greater than or less than 1.0 and includes for example, aratio of greater than 1.1, 1.2, 1.5, 1.7, 2, 3, 4, 10, 20, or a ratioless than 1, for example 0.9, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05.

In some embodiments, a biomarker can be identified by measuring thelevel of at least 1 target molecule in a test GLF or test fecal samplefrom at least one test subject having a condition or physiological stateand a control GLF or control fecal sample from at least 1 controlsubject not having the condition or physiological state; comparing thelevel of the at least 1 target molecule in the test GLF or test fecalsample with the level of the at least 1 target molecule in the controlGLF or control fecal sample, wherein a significant difference in thelevel of the at least 1 target identifies a biomarker. Some embodimentsinclude measuring and comparing a plurality of target molecules in atest GLF or test fecal sample from plurality of test subjects having acondition or physiological state and a control GLF or control fecalsample from plurality of control subjects not having a condition orphysiological state. In some embodiments, at least 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 targetmolecules can be measured and compared. In some embodiments, a GLF orfecal sample can be obtained from at least 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 test subjects. Insome embodiments, a GLF or fecal sample can be obtained from at least 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,or 100 control subjects. In some embodiments, a significant differencein the level of a target molecule in a test GLF or a test fecal samplecompared to a control GLF or a control fecal sample can be astatistically significant.

Kits

Some embodiments of the methods and compositions provided herein relateto kits for detecting a target molecule in a GLF or fecal sample,determining the presence or absence of a target molecule in a GLF orfecal sample, quantifying a target molecule in a GLF or fecal sample, oridentifying a target molecule in a GLF or fecal sample. Some such kitscan include a lavage composition for oral administration to a subject.In some embodiments, the lavage fluid can include an ingredient such aspolyethylene glycol, magnesium sulfate, sodium sulfate, potassiumsulfate, magnesium citrate, and bisacodyl. In some embodiments, thelavage fluid can include polyethylene glycol with an electrolytesolution, optionally also including bisacodyl, or ascorbic acid (e.g.,GOLYTELY, HALFLYTELY, NULYTELY, MOVIPREP). In some embodiments, thelavage fluid can include phosphate salts (e.g. FLEET'S PHOSPHO-SODA). Insome embodiments, the lavage fluid can include sulfate salts such assodium sulfate, magnesium sulfate, or potassium sulfate (e.g., SUPREP).In some embodiments, the lavage fluid can include magnesium citrate. Insome embodiments, the lavage fluid can include sodium picosulfate.

In some embodiments, a kit can also include a vessel for collecting aGLF and/or fecal sample from a subject. A vessel for collecting a GLFcan include an insert for a toilet which captures the GLF or fecalsample and the like. In some embodiments, the vessel can include amaterial to stabilize and/or preserve a target molecule, such as one ormore isolated protease inhibitors. In some embodiments, the vessel caninclude an agent for detecting a target molecule, determining thepresence or absence of a target molecule, quantifying a target moleculeor identifying a target molecule.

Diseases

Some embodiments of the methods and compositions provided herein relateto the diagnosis, prognosis for a particular disease. Some embodimentsinclude diseases and disorders related to the GI tract and organsassociated therewith. Example diseases include cancers of the GI tractand organs associated therewith, e.g., gastric cancer, liver cancer,pancreatic cancer. More examples of diseases include pancreatitis,pancreatic adenocarcinoma, gastrointestinal neuroendocrine tumors,gastric adenocarcinoma, colon adenocarcinoma, hepatocellular carcinoma,cholangiocarcinoma, gallbladder adenoccarcinoma, ulcerative colitis, andCrohn's disease. Some diseases relate to an inflammatory bowel disease(IBD). As used herein, the term “inflammatory bowel disease” can referto a broad class of diseases characterized by inflammation of at leastpart of the gastrointestinal tract. IBD symptoms may includeinflammation of the intestine and resulting in abdominal cramping andpersistent diarrhea. Inflammatory bowel diseases include ulcerativecolitis (UC), Crohn's disease (CD), indeterminate colitis, chroniccolitis, discontinuous or patchy disease, ileal inflammation,extracolonic inflammation, granulomatous inflammation in response toruptured crypts, aphthous ulcers, transmural inflammation, microscopiccolitis, diverticulitis and diversion colitis. More examples of diseasesinclude celiac sprue, malabsorption disorders, and other conditions ofdigestive tract, liver, pancreas, and biliary tree.

Some embodiments of the methods and compositions provided herein relateto determining the selection of a treatment (often referred to aspersonalized medicine), a subject's positive response to treatment,negative response to treatment, or lack of response to treatment. Somesuch embodiments include determining a patient's partial response to atreatment regimen. For example, the presence of a biomarker, absence ofa biomarker, or level of a biomarker can be determined in a GLF or fecalsample from a subject at a first time point. At a second time pointafter treatment has begun and/or treatment has been completed, thepresence of the biomarker, absence of the biomarker, or level of thebiomarker can be determined in a GLF or fecal sample from the subject.The difference in the presence of the biomarker, absence of thebiomarker, or level of the biomarker in the GLF or fecal sample at thesecond time point compared with the presence of the biomarker, absenceof the biomarker, or level of the biomarker in the GLF or fecal samplefrom the first time point can be indicative of the subject's positiveresponse to treatment, negative response to treatment, partial responseto treatment, or lack of response to treatment. Alternatively, subjectscan be given a treatment regimen and categorized as having a positiveresponse, negative response, partial response, or no response. Thepresence, absence, or level of a target molecule in each group ofsubjects can be determined and those target molecules having astatistically significant association with each category of response canbe identified. Some embodiments also include determining a futuretreatment regimen to be provided to a subject in view of determining thesubject's positive response, negative response, partial response, or noresponse to a former or current treatment regimen. Accordingly, a formeror current treatment regimen can be modified based on determinationsmade by the methods provided herein.

More embodiments include methods for determining a subject'sphysiological status by evaluating a plurality of biomarkers. Some suchmethods include determining the presence, absence and/or levels of aplurality of biomarkers. The presence, absence and/or levels of aplurality of biomarkers can be correlated to the likelihood of thesubject's physiological status, such as the subject's likelihood ofdeveloping a disease, and/or a subject's likely response to a treatmentregimen to treat a particular disease. In some such methods, a subject's“clinical risk score” can be determined by correlating the presence,absence and/or levels of a plurality of biomarkers to determine thelikelihood that a subject has a disease or will develop a disease (see,e.g., Soonmyung P. et al., (2004) New Eng. J. of Medicine 351:2817-2826;and Cho C. S. et al., (2008) J. Am. Coll. Surg. 206:281-291,incorporated by reference herein in their entireties).

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention.

EXAMPLES Example 1—Proteomic Analysis of Sulfate-Based GLF

In this analysis, the ability of a sulfate-based GLF to supportproteomic analysis was assessed. To identify target molecules in a GLFobtaining using a sulfate-based lavage composition, SUPREP wasadministered to three human subjects, and proteins in the resultant GLFwere analyzed by mass spectroscopy. In this example, the GLF wascollected from subjects as part of a colonoscopy procedure.

Upon collection, a complete protease inhibitor tablet (ROCHE) was addedand samples were spun at 1000 rpm for 30 minutes at 4° C. Supernatantswere spun again at 14,000×g for 30 min to pellet bacteria and debris.1.8 ml of supernatant was precipitated with 6 volumes of acetonefollowed by extraction with an equal volume of chloroform followed byseparation on a C-2 reverse phase SPE column (Sep-Pak, Waters). Thecolumn was washed with 3 column volumes each of 0.1% trifluoroaceticacid (TFA), 10%, 20%, and 30% acetonitrile (ACN) in 0.1% TFA, and elutedwith 3 column volumes of 60% ACN in 0.1% TFA. Samples were dried bycentrifugal lyophilization, resuspended in 100 μl of 50 mM ammoniumbicarbonate/10 mM tris (2-carboxyethyl) phosphine and digested with 2 μlof 10 mM sequencing-grade trypsin (Promega, Madison, WI).

Data were acquired on an LTQ-Orbitrap mass spectrometer using input froman LC system. The A solvent contained 3% of B and 0.2% formic acid inwater. The B solvent contained 3% of A and 0.2% formic acid inacetonitrile. Solvents were HPLC grade from Fisher. For a 120 min run,the starting solvent was 5% B and remains for 7 min. The gradient waschanged to 10% by 13 min, 40% by 83 min, 90% by 103 min, then reducedfrom 90% to 5% at 111 min. It was then re-equilibrated for the nextinjection. Three injections were performed for each sample forrepeatability determination.

The MS was scanned (Orbitrap) over the mass range from 400 m/z to 2000m/z every second while the LTQ (Trap) acquired up to 5 MSMS (peptidesequence) spectra in parallel. Data were acquired using the standardThermo Xcalibur software. MS data (Orbitrap) was stable to 2-3 ppm and abackground ion was used for mass drift assessment. MSMS data (LTQ) wasmeasured to approximately 0.6 Da but the parent mass was acquired fromthe low ppm Orbitrap data. Peptides were eluted from a C18 LC columnusing triplicate injections to ensure reliability and repeatability ofthe data. A search file was created from the triplicate injections fromeach lavage preparation (patient sample) and converted into a MGF(Mascot Generic Format) file using a combination of Xcalibur and Mascotsoftware packages.

Database searching was done using the Mascot search engine (MatrixScience, UK) against the RefSeq database (http://www.ncbi.nlm nihgov/RefSeq/) with taxonomy specified as human (homo sapiens), a massaccuracy of 10 ppm for the parent ion (MS) and 0.6 Da for the fragmentions (MS/MS), and “no enzyme” selected. Searching without enzymespecificity was performed due to the presence of digestive enzymes inthe sample that may modify or truncate peptides being examined TheRefSeq database was supplemented by the addition of antibody sequencesthat are included in the SwissProt protein database, as these antibodysequences are not part of the standard RefSeq listing.

Higher Mascot scores indicate better proteins hits and can be correlatedto relative protein levels. A score threshold of “>40” was indicative ofa p-value significance of <0.05 as determined by the Mascot scoringsystem based on the search of this database with no enzyme specificity;a score of 40 is consistent with a p<0.01. Standard Mascot scoring wasused whereby only the highest score was added for each peptide detected,even if it was sampled during MS/MS multiple times. For all dataincluded, scores were all >40 in at least one sample per protein line.For additional confidence, the numbers of significant peptides were alsoreported and a minimum criterion of at least 2 peptides was selected.Very few had less than 3 peptides. All significant peptides countedrepresented different sequences (individual peptides) from theirrespective proteins. The score and numbers of significant peptides arereported in the format x/y where x is the score and y the number ofsignificant peptides. If a protein was not detected in a particularsample it is listed as “ND”. Proteins are reported as protein name andthe “gi” number defined by the protein database of the NCBI has beenprovided. The sequences contained in each of the “gi” numbers in theNCBI database listed throughout the present application are incorporatedherein by reference. Where a protein is named in its preprotein or othernon-mature form, the mature form of the protein is equally impliedincluding such changes as removal of signal sequences and the additionof post-translational modifications. In all cases, the protein has beennamed by its gene derived sequence to provide consistency.

Table 1 lists examples of the most abundant proteins identified in GLFfrom three separate patients defined as patients 3, 4 and 6, presentedin the format described above. As can be seen from Table 1, manyproteins can be identified from GLF and a large number of these may beassociated with the pancreas. Other proteins include DMBT1 (gi #148539840) which may be associated with colon cancer and other cancers.Antibodies and putative glycosylated proteins were also identified.

TABLE 1 Mascot score/ number of significant peptides NCBI gi # ProteinSample 3 Sample 4 Sample 6 10835000 pancreatic lipase precursor 2224/241238/13 2926/34 4506147 protease serine 2 preproprotein 665/7  46/01189/11 4506145 protease serine 1 preproprotein 239/2  64/0 1002/1129725633 regenerating islet-derived 1 alpha 231/2 132/1 802/6 precursor6679625 elastase 3B pancreatic preproprotein  852/12 1144/11 772/8236460050 elastase 3A pancreatic preproprotein 1291/17 1306/14 769/7118498350 chymotrypsin B2  945/10 244/2 724/6 15559207 elastase 2Apreproprotein  752/10 952/8 593/6 54607080 pancreatic carboxypeptidaseB1 702/9  84/1 499/4 preproprotein 50363217 serine proteinase inhibitorclade A 655/9 406/3 490/2 member 1 10280622 amylase pancreatic alpha-2Bprecursor ND ND 388/2 4505847 phospholipase A2 group IB 258/3 639/8384/5 4502085 pancreatic amylase alpha 2A precursor  95/1 193/3 365/24502997 carboxypeptidase A1 precursor 454/4  88/1 349/5 62526043chymotrypsin C preproprotein 696/9 440/3 343/4 148539840 deleted inmalignant brain tumors 1  88/1 101/1 280/3 isoform a precursor (DMBT1)31377806 polymeric immunoglobulin receptor 566/7 ND 279/3 precursor41152086 serine (or cysteine) proteinase inhibitor ND 269/3 276/2 cladeB (ovalbumin) member 6 148539842 deleted in malignant brain tumors 1 NDND 275/3 isoform b precursor 4507149 superoxide dismutase 1 soluble ND 87/1 253/3 113584 RecName: Full = Ig alpha-1 chain C region  940/10 53/1 204/2 125145 RecName: Full = Ig kappa chain C region 659/9 106/1180/1 98986445 carcinoembryonic antigen-related cell ND 219/3 135/0adhesion molecule 5 preproprotein 218512088 RecName: Full = Ig alpha-2chain C region 886/9 ND ND 119395750 keratin 1 ND 499/7 ND 55956899keratin 9 ND 395/3 ND

In another experiment, SUPREP was administered to a subject according tothe manufacturer's guidelines and the resultant GLF was self-collectedby the subject into a collection container placed in the toiletimmediately prior to colonoscopy. The proteome of the GLF was analyzedby MS as described above. The results showing the Mascot scores for themost abundant species present are summarized in Table 2. The resultsindicated some urinary contamination. A similar proteomic profile wasobserved for a sample collected subsequently during colonoscopy. Table 2shows that many different proteins were identified in GLF collected by asubject. Identified proteins included DMBT1, pancreatic proteins andantibodies, consistent with data in Table 1.

TABLE 2 Mascot score/ number of significant peptides NCBI gi # ProteinSample 25 148539842 deleted in malignant brain tumors 1 isoform b1184/13 precursor 119395750 keratin 1 742/9 10835000 pancreatic lipaseprecursor 538/8 113584 RecName: Full = Ig alpha-1 chain C region 506/631377806 polymeric immunoglobulin receptor precursor 474/5 98986445carcinoembryonic antigen-related cell adhesion 424/5 molecule 5preproprotein 125817 RecName: Full = Ig kappa chain V-III region HAH;382/5 Flags: Precursor 125797 RecName: Full = Ig kappa chain V-IIIregion SIE 341/5 54607080 pancreatic carboxypeptidase B1 preproprotein340/5 236460050 elastase 3A pancreatic preproprotein 328/4 125145RecName: Full = Ig kappa chain C region 327/5 4502027 albuminpreproprotein 319/3 33456 immunoglobulin M chain 239/3 118498350chymotrypsin B2 238/3 125811 RecName: Full = Ig kappa chain V-III regionVG; 237/3 Flags: Precursor 123843 RecName: Full = Ig heavy chain V-IIIregion VH26; 219/3 Flags: Precursor 157266300 membrane alanineaminopeptidase precursor 213/3 563454 Ig heavy chain (VH4) V region(VDJ) 206/3 125788 RecName: Full = Ig kappa chain V-II region TEW 202/34506147 protease serine 2 preproprotein 177/3 125809 RecName: Full = Igkappa chain V-III region CLL; 149/3 AltName: Full = Rheumatoid factor;Flags: Precursor

The foregoing analyses demonstrate that a large number of targetmolecules can be detected in samples obtained using a sulfate-based GLF.

Example 2—Proteomic Analysis of Polyethylene Glycol Based GLF

In this analysis, the ability of a polyethylene glycol based GLF tosupport proteomic analysis was assessed. To identify target molecules ina GLF obtaining using a polyethylene-based lavage composition, apolyethylene glycol-based lavage composition was administered to twohuman subjects, and proteins in the resultant GLF were analyzed by massspectrometry as described in Example 1. Removal of the polyethyleneglycol was largely achieved by chloroform extraction of the lavagefluid. Many different proteins were identified in the GLFs from thesesubjects administered a polyethylene glycol-based lavage composition.Examples of the most abundant identified proteins identified, which areconsistent with those observed in previous tables, are presented inTable 3.

TABLE 3 Mascot score/ number of significant peptides NCBI gi# ProteinSample 1 Sample 5 98986445 carcinoembryonic antigen-related celladhesion  767/10 127/1 molecule 5 preproprotein 4502085 pancreaticamylase alpha 2A precursor 619/8 ND 1684927 immunoglobulin light chain586/6 152/1 50363217 serine proteinase inhibitor clade A member 1 550/4268/2 40254482 salivary amylase alpha 1A precursor 548/6 ND 298351713RecName: Full = Ig lambda-1 chain C regions 501/6 139/1 4507725transthyretin precursor 477/5 ND 236460050 elastase 3A pancreaticpreproprotein 432/5  84/0 4502997 carboxypeptidase A1 precursor 412/5106/1 113584 RecName: Full = Ig alpha-1 chain C region 404/4 214/34885165 cystatin A 352/4  66/1 40255013 carcinoembryonic antigen-relatedcell adhesion 349/3 ND molecule 6 (non-specific cross reacting antigen)4506147 protease serine 2 preproprotein 326/5 434/6 125145 RecName: Full= Ig kappa chain C region 291/4 265/5 218512088 RecName: Full = Igalpha-2 chain C region 272/3 ND 121039 RecName: Full = Ig gamma-1 chainC region 265/3 ND 54607080 pancreatic carboxypeptidase B1 preproprotein263/2 ND

In another experiment, a PEG-based lavage composition was administeredto a subject and the subject self-collected the resultant GLF into acollection container placed in the toilet immediately prior tocolonoscopy. The proteome of the GLF was analyzed by MS as describedherein. Many different proteins were identified in the self collectedGLF sample. Examples of the most abundant identified proteins, and thecorresponding Mascot scores and numbers of significant peptides for eachprotein are listed in Table 4. The more extensive protein list showedevidence of urinary contamination. A similar proteomic profile wasobserved for a sample collected subsequently during colonoscopy.

TABLE 4 Mascot score/ number of significant peptides NCBI gi # ProteinSample 26 50363217 serine proteinase inhibitor clade A 406/3 member 14885165 cystatin A 294/3 4502027 albumin preproprotein 287/3 55956899keratin 9 287/4 4506147 protease serine 2 preproprotein 227/3 4502085pancreatic amylase alpha 2A precursor 217/3

The proteomes of GLFs resultant from the administration of sulfate-basedlavage compositions and either collected during as part of a colonoscopyprocedure or self-collected by a subject were compared. The Mascotscores and number of significant peptides for the most abundant proteinsare summarized in Table 5. While there was a close correlation betweenthe two proteomes observed, different isoforms were identified for atleast two proteins. The selection of different isoforms may be a resultof the collection of the sequence data during MS/MS and the searchengine. There were fewer proteins detected in the self collected samplewhich was more dilute than that collected during the colonoscopy.

TABLE 5 Mascot score/ number of significant peptides Subject Colonoscopycollected NCBI gi # Protein collected sample sample 148539842 deleted inmalignant brain tumors See isoform a 1184/13 1 isoform b precursor148539840 deleted in malignant brain tumors 417/4 See isoform b 1isoform a precursor 119395750 keratin 1 159/2 742/9 10835000 pancreaticlipase precursor 3719/37 538/8 113584 RecName: Full = Ig alpha-1 chain C958/9 506/6 region 31377806 polymeric immunoglobulin 469/4 474/5receptor precursor 98986445 carcinoembryonic antigen-related  74/1 424/5cell adhesion molecule 5 preproprotein 125817 RecName: Full = Ig kappachain V- ND 382/5 III region HAH; Flags: Precursor 125797 RecName: Full= Ig kappa chain V- ND 341/5 III region SIE 54607080 pancreaticcarboxypeptidase B1 1389/17 340/5 preproprotein 236460050 elastase 3Apancreatic 2268/27 328/4 preproprotein 125145 RecName: Full = Ig kappachain C 734/7 327/5 region 118498350 chymotrypsin B2 See chymotrypsin238/3 B1 118498341 chymotrypsin B1 881/7 See chymotrypsin B2 62526043chymotrypsin C preprotein 1002/10 ND 125811 RecName: Full = Ig kappachain V- ND 237/3 III region VG; Flags: Precursor 1684927 immunoglobulinlight chain 371/4 221/2 123843 RecName: Full = Ig heavy chain V- 181/2219/3 III region VH26; Flags: Precursor 157266300 membrane alanineaminopeptidase 544/5 213/3 precursor 125788 RecName: Full = Ig kappachain V- ND 202/3 II region TEW 4502085 pancreatic amylase alpha 2A4258/49 ND precursor 10280622 amylase pancreatic alpha-2B 3916/45 NDprecursor 6679625 elastase 3B pancreatic 1955/22 ND preproprotein4506147 protease serine 2 preproprotein 1442/14 ND 4502997carboxypeptidase A1 precursor 1168/14 ND 15559207 elastase 2Apreproprotein 959/5 ND 217416390 carboxypeptidase A2 (pancreatic) 811/10 ND precursor 29725633 regenerating islet-derived 1 alpha 714/8ND precursor 218512088 RecName: Full = Ig alpha-2 chain C 663/6 NDregion 7669492 glyceraldehyde-3-phosphate 593/5 ND dehydrogenase 4506145protease serine 1 preproprotein 551/6 ND 157266300 membrane alanineaminopeptidase 544/5 ND precursor 298351713 RecName: Full = Ig lambda-1chain 322/4 ND C regions 119220569 zymogen granule membrane 304/3 NDglycoprotein 2 isoform 1 51593090 mucin 13 epithelial 293/3 NDtransmembrane 125807 RecName: Full = Ig kappa chain V- 288/3 ND IIIregion GOL; AltName: Full = Rheumatoid factor 10334859 creatine kinasemitochondrial 1B 265/3 ND precursor

The foregoing analyses demonstrate that a large number of targetmolecules can be detected in samples obtained using a polyethyleneglycol based GLF.

Example 3—Proteomic Analysis of Magnesium Citrate Based GLF

In this analysis, the ability of a magnesium citrate based GLF tosupport proteomic analysis was assessed. To identify target molecules ina GLF from a human subject administered a magnesium citrate-based lavagecomposition, a magnesium citrate-based lavage composition wasadministered to a subject; the GLF was collected from subject as part ofa colonoscopy procedure. The proteome of the GLF was analyzed by massspectroscopy as described in Example 1. Many different proteins wereidentified in the GLF. Examples of the most abundant identified proteinsare listed in Table 6. Many of the identified proteins were detectedwith different colonoscopy preparations suggesting that the proteome isnot dependent on the bowel preparation used.

TABLE 6 Mascot score/ number of significant peptides NCBI gi # ProteinSample 27 4502085 pancreatic amylase alpha 2A precursor 2977/31 10280622amylase pancreatic alpha-2B precursor 2891/30 40254482 salivary amylasealpha 1A precursor 2472/26 50363217 serine proteinase inhibitor clade Amember 1 1316/12 236460050 elastase 3A pancreatic preproprotein 1299/1315559207 elastase 2A preproprotein 1109/11 6679625 elastase 3Bpancreatic preproprotein  987/10 29725633 regenerating islet-derived 1alpha precursor 577/6 4507725 transthyretin precursor 570/7 4506147protease serine 2 preproprotein 521/6 58331211 elastase 2B preproprotein498/4 157266292 intestinal alkaline phosphatase precursor 491/7 113584RecName: Full = Ig alpha-1 chain C region 338/3 125145 RecName: Full =Ig kappa chain C region 337/4 98986445 carcinoembryonic antigen-relatedcell adhesion 329/3 molecule 5 preproprotein 218512088 RecName: Full =Ig alpha-2 chain C region 293/3 41152086 serine (or cysteine) proteinaseinhibitor clade B 293/4 (ovalbumin) member 6 4506145 protease serine 1preproprotein 267/3 4502997 carboxypeptidase A1 precursor 146/3

The foregoing analysis demonstrates that a large number of targetmolecules can be detected in samples obtained using a magnesium citratebased GLF.

Example 4—Detection of IgA-1 and IgA-2 Antibodies in Samples ObtainedUsing a GLF in Combination with SSL-7 Enrichment

In this analysis, the ability of a samples obtained using a GLF incombination with SSL-7 enrichment to detect IgA-1 and IgA-2 antibodieswas assessed. To identify target molecules in a GLF with affinity toStaphylococcus aureus superantigen-like protein 7 (SSL-7), asulfate-based lavage composition (SUPREP) was administered to humansubjects, and proteins were enriched in each GLF using SSL-7 affinitybeads. The GLF was collected from subjects as part of a colonoscopyprocedure.

SSL-7 affinity beads were used to isolate IgA-1 and IgA-2 specifically.20 μl of SSL-7 agarose (Invitrogen, San Diego, Calif.) was added to 1 mlof sample and incubated overnight on a roller at 4° C. Tubes were spunat 1,000×g for 2 minutes to pellet beads and supernatant discarded.Beads were washed 4× with 1× phosphate buffered saline and eluted with20 μl of 100 mM glycine, pH 2.7 in a shaker for 1 hr at 600 rpm and 37°C. Eluted antibodies were diluted with 60 μl of 100 mM ammoniumbicarbonate/10 mM tris (2-carboxyethyl) phosphine and digested with 2 μlof sequencing grade trypsin (Promega, Madison, Wis.). Mass spectrometryand database searches were performed as described above. The mostabundant identified proteins present in GLF with affinity to SSL-7 andtheir corresponding Mascot scores are summarized in Table 7. As has beenobserved in prior examples, antibodies were present in the GLF andenrichment and analysis of these is possible using the affinityreagents, thus allowing the specific analysis of this subproteome in theGLF. The most abundant antibodies were IgAs. IgAs are consistentlyreported to be present in the intestinal tract.

TABLE 7 Mascot score/ number of significant peptides NCBI gi # ProteinSample 12 Sample 13 113584 RecName: Full = Ig alpha-1 3084/29 2477/24chain C region 31377806 polymeric immunoglobulin 3065/36  898/13receptor precursor 218512088 RecName: Full = Ig alpha-2 chain 2736/242225/18 C region 125145 RecName: Full = Ig kappa chain 477/4 542/6 Cregion 21489959 immunoglobulin J chain 403/4 308/2 298351715 RecName:Full = Ig lambda-3 352/3 ND chain C regions 298351713 RecName: Full = Iglambda-1 317/3 581/6 chain C regions 1684927 immunoglobulin light chainND 636/6

The foregoing analysis demonstrates that IgA antibodies can be detectedin samples obtained using a GLF in combination with SSL-7 enrichment.

Example 5—Detection of IgA and IgM in Samples Obtained using a GLF inCombination with ProteinL Enrichment

In this analysis, the ability of samples obtained using a GLF incombination with Protein L enrichment to detect IgA and IgM antibodieswas assessed. To identify target molecules in a GLF with affinity toProtein L, a sulfate-based lavage composition (SUPREP) was administeredto human subjects, and proteins were enriched in each GLF using ProteinL affinity beads. The GLF was collected from subjects as part of acolonoscopy procedure.

Protein L affinity beads were used to isolate antibodies containingkappa light chains. 20 μl of Protein L agarose (Santa CruzBiotechnology, Santa Cruz, Calif.) was added to 1 ml of sample andincubated overnight on a roller at 4 C. Tubes were spun at 1,000×g for 2minutes to pellet beads and supernatant discarded. Beads were washed 4times with 1× phosphate buffered saline and eluted with 20 μl of 100 mMglycine, pH 2.7 in a shaker for 1 hr at 600 rpm and 37° C. Elutedantibodies were diluted with 60 μl of 100 mM Ammonium bicarbonate/10 mMtris (2-carboxyethyl) phosphine and digested with 2 μl of sequencinggrade trypsin (Promega, Madison, Wis.). The most abundant identifiedproteins present in GLF with affinity to Protein L and theircorresponding Mascot scores and numbers of significant peptides aresummarized in Table 8. As expected, IgA and associated chains fromantibodies were again detected. As the Protein L is not totally specificfor IgA antibodies, an IgM antibody (gi# 193806374) was also detected.

TABLE 8 Mascot score/ number of significant peptides NCBI gi # ProteinSample 9 113584 RecName: Full = Ig alpha-1 chain C region 1183/1431377806 polymeric immunoglobulin receptor 1149/14 precursor 218512088RecName: Full = Ig alpha-2 chain C region  985/11 125145 RecName: Full =Ig kappa chain C region 654/8 193806374 RecName: Full = Ig mu chain Cregion 407/5 187950123 immunoglobulin heavy chain variable 289/3 region21489959 immunoglobulin J chain 249/2

The foregoing analysis demonstrates that IgA and IgM antibodies can bedetected in samples obtained using a GLF in combination with ProteinLenrichment.

Example 6—Detection of Proteins of Bacterial Origin in Samples ObtainedUsing a GLF

In this analysis, the ability of a GLF to facilitate detection ofproteins of bacterial origin was assessed. To identify target moleculesassociated with bacteria in a GLF, a sulfate-based lavage composition(SUPREP) was administered to two human subjects; the resultant GLF wascollected from the subjects as part of a colonoscopy procedure. SuperOptimal Broth (SOB) media was inoculated with 100 μl from each GLF andincubated overnight at 37° C. and 220 rpm shaking. Pellets were lysed ina bead-beater in 8M urea and lysates were diluted to 2 M urea in 50 mMammonium bicarbonate/10 mM tris (2-carboxyethyl) phosphine and digestedwith sequencing grade trypsin (Promega, Madison, Wis.). Data wereacquired on the Orbitrap MS system using 120 mins runs as describedearlier. A MGF search file was created and searched with the Mascotsearch engine (Matrix Science, UK) against the RefSeq database(http://www.ncbi.nlm nih.gov/RefSeq/) with the taxonomy specified asEubacteria with a mass accuracy of 10 ppm for the parent ion (MS) and0.6 Da for the fragment ions (MS/MS). The most abundant identifiedproteins present in GLF associated with bacteria and their correspondingMascot scores and numbers of significant peptides are summarized inTable 9. In the sample shown, the bacterium that was cultured wasEscherichia coli. Other samples show different bacteria showing that thelavage fluid still retains some of the gut bacteria.

TABLE 9 Mascot score/ number of significant peptides NCBI gi # ProteinBacterial isolate 15834378 chaperonin GroEL [Escherichia coli] 1294/315803852 elongation factor Tu [Escherichia coli] 1244/7 157159481Molecular chaperone DnaK  683/3 [Escherichia coli] 123444102 elongationfactor Tu [Yersinia  518/3 enterocolitica subsp. enterocolitica 8081]

The foregoing analysis demonstrates that proteins of bacterial origincan be detected in samples obtained using a GLF.

Example 7—Proteomic Analysis of Combined Samples from Several Subjects

In order to further facilitate the identification of a large number oftarget proteins detectable in samples obtained using a GLF, the searchfiles generated from the data acquired individually from 12 subjectswere concatenated into a single search file and searched using thepreviously specified parameters for the Orbitrap data. Many proteinswere analyzed in various GLF samples and proteins selected that(predominantly) had at least 3 unique significant peptides detected withthresholds of p<0.05 (Mascot score approximately 41). Only 3 listedproteins (gi's 5031863, 6466801 and 115430223) had less than 3significant peptides but these had Mascot scores of approximately 400,well above the 95% confidence level for protein identification. Proteinsidentified in this combined analysis are listed in Table 10 along withreported origins of particular proteins and reported associated cancers.Table 10 also lists SEQ ID NO.s. for identified peptides that had Mascotscores of 40 or greater for each unique identified protein. Manyidentified proteins have been reported to be present in pancreaticjuice. References listed in Table 10 are provided in this application.

TABLE 10 Mascot score/ SEQ ID NO.s number of of peptides with Origin ofsignificant Mascot scores detected Presence in NCBI gi# Detected proteinpeptides >40 protein pancreatic juice Associated cancer References10835000 pancreatic lipase 6919/75  1-77 pancreas Yes pancreatic Friess(2003) precursor 4502085 pancreatic amylase 5766/60  78-137 pancreas Yesgastric Kang (2010) alpha 2A precursor 10280622 amylase, 5332/55 78-83,85-100, pancreas Yes liver Koyama (2001) pancreatic, alpha- 102, 103,105-108, 2B precursor 110-130, 133, 136-141 40254482 salivary amylase4712/50 78-83, 86-90, pancreas/ Yes lung Tomita (1989) alpha 1Aprecursor 93-99, 102, 105-107, salivary gland 110-116, 118-126, 128,129, 131, 133-135, 139, 140, 142, 143 236460050 elastase 3A, 4267/49144-193 pancreas Yes lung Shimada (2002) pancreatic preproprotein6679625 elastase 3B, 4123/44 145-147, 151-153, pancreas Yes pancreaticGao (2010) pancreatic 155, 157, preproprotein 161-163, 165, 166, 168,170, 172-176, 178, 179, 181, 183, 185, 186, 188, 191, 194-211 4506147protease, serine, 2 3427/33 212-247 pancreas Yes pancreatic Gao (2010)preproprotein 118498350 chymotrypsin B2 2621/27 248-274 pancreas Yespancreatic Miao (2008) 118498341 chymotrypsin B1 2527/26 248-256,258-274 pancreas Yes general Miao (2008) 50363217 serine proteinase2443/27 275-304 pancreas/liver Yes general Normandin inhibitor, clade A,(2010), Sato member 1 (2004), Zhou (2000) 15559207 elastase 2A 2351/25305-331 pancreas Yes pancreatic Akakura (2001) preproprotein Yamamura(1989) 54607080 pancreatic 2143/24 332-355 pancreas Yes liver Matsugi(2007) carboxypeptidase B1 preproprotein 4506145 protease, serine, 12135/21 215, 217, 219, pancreas Yes pancreatic preproprotein 221, 222,225, 228, 229, 231, 232, 236, 238-240, 242, 245, 356-361 148539844deleted in 2129/23 362-385 epithelial, Yes pancreatic, Sasaki (2002),malignant brain pancreas brain, Kuramitsu (2006) tumors 1 isoform clung, precursor colon, gastric 113584 RecName: Full = Ig 2065/19 386-405antibody- Yes alpha-1 chain C heavy chain region IgA 4502997carboxypeptidase 2026/25 334, 406-429 pancreas Yes pancreatic Matsugi(2007) A1 precursor 29725633 regenerating islet- 1799/17 430-444pancreas Yes liver Cavard (2006) derived 1 alpha precursor 31377806polymeric 1783/19 445-465 epithelial Yes endometrial DeSouza (2005)immunoglobulin receptor precursor 218512088 RecName: Full = Ig 1779/16386-388, 390-392, antibody- Yes alpha-2 chain C 397, 399-404, heavychain region 466-469 157266300 membrane alanine 1581/16 470-486 smallintestine Yes breast Liang (2006) aminopeptidase precursor 119395750keratin 1 1491/15 487-501 epithelial 62526043 chymotrypsin C 1407/16502-517 pancreas Yes liver Wang (2011) preproprotein 98986445carcinoembryonic 1140/11 518-528 epithelial Yes pancreatic, Sato (2004),Van antigen-related cell colon Gisbergen (2005) adhesion molecule 5preproprotein 217416390 carboxypeptidase 1129/11 529-539 pancreas Yespancreatic Matsugi (2007) A2 (pancreatic) precursor 4505847phospholipase A2 1124/13 540-552 pancreas Yes colon, Belinsky (2007),group IB prostate Sved (2004) 125145 RecName: Full = Ig 1121/14 553-566antibody- Yes kappa chain C light chain region 7669492 glyceraldehyde-3-1106/11 567-578 epithelial/bacterial colon Egea (2007), phosphate Shin(2009) dehydrogenase 58331211 elastase 2B 1083/11 305, 307-310, pancreaspreproprotein 312-314, 318, 324, 328, 331 105990514 filamin B, beta1041/7  579-586 multiple cell prostate Harding (2006) (actin bindingtypes protein 278) 4507725 transthyretin  952/11 587-597 Liver/serum Yescolon Fentz (2007) precursor protein 55956899 keratin 9 945/9 598-607epithelial 170296790 mesotrypsin 907/8 214, 215, 218, pancreas breastHockla (2010) isoform 1 219, 221, 224, preproprotein 226, 233, 60810835248 regenerating islet- 903/9 430, 431, 433-437, pancreas Yespancreatic Cui (2010) derived 1 beta 441 precursor 41152086 serine (orcysteine) 902/9 609-617 keritinocytes, Yes colon Krasnov (2009)proteinase muscle, lung, inhibitor, clade B liver, (ovalbumin), pancreasmember 6 1684927 immunoglobulin 889/8 618-625 light chain 4505605pancreatitis- 848/9 626-634 pancreas Yes pancreatic Rosty (2002)associated protein precursor 13489087 serine (or cysteine) 842/8 635-642keritinocytes, Yes pancreatic Sato (2004) proteinase muscle, lung,inhibitor, clade B liver, (ovalbumin), pancreas member 1 226529917triosephosphate 760/7 643-649 multiple cell Yes breast Tamesa (2009)isomerase 1 types isoform 2 298351713 RecName: Full = Ig 760/8 618-623,625, antibody- lambda-1 chain C 650 light chain regions 47132620 keratin2 713/7 487, 495, 651-655 epithelial 5080756 Human Fc gamma 706/4656-659 BP [AA 1-2843] 195972866 keratin 10 676/5 660-664 epithelialLiver, Yang (2008), pancreatic Xiao (2010) 4502027 albumin 642/4 665-668preproprotein 40255013 carcinoembryonic 614/5 522, 524, 669-671epithelial Yes colon Van Gisbergen antigen-related cell (2005) adhesionmolecule 6 (non-specific cross reacting antigen) 125819 RecName: Full =Ig 611/6 672-677 antibody- leukemia Kipps (1988) kappa chain V-III lightchain region HIC; Flags: Precursor 154146262 Fc fragment of IgG 607/4656-659 prostate Gazi (2008) binding protein 157266292 intestinalalkaline 605/7 678-684 small intestine liver Yamamoto phosphatase (1984)precursor 50845388 annexin A2 isoform 1 604/5 685-689 multiple cellliver Mohammad types (2008) 50659080 serpin peptidase 599/4 690-693liver melanoma Wang (2010) inhibitor, clade A, member 3 precursor51593090 mucin 13, 575/5 694-698 colon GI cancer Maher (2011) epithelialtransmembrane 119220569 zymogen granule 567/4 699-702 pancreas Yesmembrane glycoprotein 2 isoform 1 125817 RecName: Full = Ig 554/6672-677 antibody- leukemia Kipps (1988) kappa chain V-III light chainregion HAH; Flags: Precursor 151301154 mucin 6, gastric 544/3 703-70510334859 creatine kinase, 533/5 706-711 mitochondria tissue Bark (1980)mitochondrial 1B damage precursor 223099 Ig Aalpha1 Bur 520/4 388, 391,467-469 4504919 keratin 8 506/6 712-717 epithelial skin Yamashiro (2010)119703753 keratin 6B 504/4 488, 653, 654, epithelial breast Millar(2009) 718 121039 RecName: Full = Ig 501/4 719-722 antibody- gamma-1chain C heavy chain region 4507149 superoxide 498/5 723-727 epithelial/Yes multiple Pham (2009) dismutase 1, mitochondria soluble 223942069enterokinase 497/3 728-731 small intestine multiple Vilen (2008)precursor 153070262 meprin A alpha 497/4 732-735 small intestine colonLottaz (1999) 157364974 sucrase-isomaltase 480/3 736-738 small intestinecolon Gu (2006) 125797 RecName: Full = Ig 478/5 672-676, 739 antibody-kappa chain V-III light chain region SIE 38455402 lipocalin 2 463/4740-743 Epithelial, Yes pancreatic, Sato (2004), Lin many cell breast,(2011), types endometrial, Mahadevin prostate (2011) 167857790orosomucoid 1 446/6 744-750 serum/acute precursor phase protein 5031839keratin 6A 441/3 653, 654, 718 epithelial breast Millar (2009) 32313593olfactomedin 4 437/4 751-754 small intestine, pancreatic, Kobayashiprecursor colon, colon (2007), Koshida pancreas (2007) 125803 RecName:Full = Ig 432/5 672-676 antibody- kappa chain V-III light chain regionWOL 10835063 nucleophosmin 1 414/5 755-759 multiple cell liver,Kuramitsu isoform 1 types others (2006), Grisendi (2006) 75707587immunoglobulin 414/4 673, 674, 676, antibody- light chain variable 760light chain region 5031863 galectin 3 binding 414/2 761, 762 multiplecell Yes colon Bresalier (2004), protein types Kim (2011) 187960098medium-chain acyl- 407/3 763-765 mitochondrial/ CoA bacterialdehydrogenase isoform b precursor 4503143 cathepsin D 407/4 766-769multiple cell breast Wolf (2003) preproprotein types 106507261pancreatic lipase- 393/2 1, 5, 770 pancreas Yes related protein 2223718246 plastin 1 393/3 771-774 small pancreatic Terris (2002)intestine, colon, kidney 4885165 cystatin A 384/4 775-778 macrophagescolon Kupio (1998), Kos (2000) 6466801 intestinal mucin 3 379/1 779epithelial pancreatic Park (2003) 115430223 galectin 3 377/2 780, 781multiple cell pancreatic Jiang (2008) types 19923195 carcinoembryonic375/4 518, 670, 782, epithelial Yes multiple Gerstel (2011)antigen-related cell 783 adhesion molecule 1 isoform 1 precursor19923748 dihydrolipoamide 362/3 784-786 mitochondria S-succinyltransferase (E2 component of 2-oxo-glutarate complex) 193806374RecName: Full = Ig 361/4 787-790 antibody- mu chain C region heavy chain16306550 selenium binding 361/3 791-793 ovarian, Huang (2006), protein 1uterine, Zhang (2010), gastric, Zhang (2011), esophageal Silvers (2010)33456 immunoglobulin M 353/4 794-797 antibody- chain heavy chain 125811RecName: Full = Ig 319/3 760, 798 799 antibody- kappa chain V-III lightchain region VG; Flags: Precursor 123843 RecName: Full = Ig 219/3 796,800, 801 antibody- heavy chain V-III heavy chain region VH26; Flags:Precursor 563454 Ig heavy chain 206/3 802-804 antibody- (VH4) V regionheavy chain (VDJ)

The foregoing analysis demonstrates that a large number of proteins canbe detected in samples obtained using a GLF.

Example 8—Proteomic Analysis of Fecal Samples

In this analysis, the ability of fecal samples to support proteomicanalysis was assessed. To identify target molecules in a fecal sample,no lavage composition was administered to a human subject. A fecalsample was collected from the subject during normal defecation using acollection container placed in the toilet. A small amount of the fecal(stool) sample was homogenized in 0.1% TFA and then centrifuged at13000×g. The protein was precipitated with 6 volumes of acetone,resuspended in 0.1% TFA, extracted with an equal volume of chloroform,and then processed in a SPE column as described in Example 1. The mostabundant identified proteins and their corresponding Mascot scores andnumber of significant peptides are summarized in Table 11. Proteins,largely with likely pancreatic origin, were detected in the sampleindicating that, after discovery in GLF, stool is a source for thedetection of these biomarker proteins. However, the sample did alsocontain a number of other non-human materials that make the analysismuch more limiting, especially for discovery of biomarkers.

TABLE 11 Mascot score/ number of significant peptides NCBI gi # ProteinSample Fecal 1 119395750 keratin 1 549/7 15559207 elastase 2Apreproprotein 546/5 236460050 elastase 3A pancreatic 532/4 preproprotein55956899 keratin 9 444/5 125145 RecName: Full = Ig kappa chain C 396/5region 4506147 protease serine 2 preproprotein 344/4 4506145 proteaseserine 1 preproprotein 320/5 118498350 chymotrypsin B2 298/3 6679625elastase 3B pancreatic 286/3 preproprotein

The foregoing analysis demonstrates that a large number of targetmolecules can be detected in fecal samples.

Example 9—Detection of Glycans in Samples Obtained Using a GLF

In this analysis, the ability of glycans to be detected in samplesobtained in samples using a GLF was assessed. To identify and analyzetarget molecules, including glycans, in a GLF, GLF was collected from ahuman subject. 1.8 mL GLF was added to 12 mL ice cold acetone andincubated for one hour to pellet the protein. The sample was centrifugedat 12,000×g for 15 minutes and the acetone removed. After washing withice cold acetone, the pellet was resuspended in 0.1% TFA and passedthrough a 5 mL, syringe style, SepPak C2 column Proteins were elutedwith 60% acetonitrile/40% 0.1% TFA. After removal of the solvent undervacuum, the protein fraction was redissolved in 100 μL 50 mM ammoniumbicarbonate and deglycosylated with 2 μL PNGaseF overnight at 37° C. ina shaker. After quenching with 1 mL 0.1% TFA, the glycans were collectedas the flow through fraction from a 1 mL, syringe style, SepPak C18column using a vacuum manifold. The dried glycans were labeled with4-ABEE (ethyl 4-aminobenzoate) by reductive amination by adding 25 μLderivatizing solution (90:10 MeOH:HAc containing 35mM ABEE and 100 mM2-PB) to the dried glycans, and incubating at 65° C. for 2 hours. ExcessABEE was removed by adding 1 mL ethyl ether, vortexing and discardingthe ether. After a second ether extraction, the sample was briefly putin the SpeedVac to remove any residual ether. The labeled glycans werethen run on the HPLC and eluted between 20-25% acetonitrile from anAgilent C8 reverse phase column. This fraction was vacuum-dried,redissolved in 50 μL of 0.1% TFA and run on the Waters Q-TOF LC-ESI-MSfor glycan analysis. The derivatized glycans eluted from the C18 reversephase column on the Q-TOF MS at about 20-25% acetonitrile in 0.2% formicacid. The mass spectrometer was scanned in MS-only mode from m/z150-2000 every second to acquire the derivatized glycan profile data.FIG. 1 summarizes these results, and depicts a graph of the relativeabundance of various glycoprotein derived glycan structures present in afraction of a gastrointestinal lavage fluid. As shown in FIG. 1, someglycoprotein derived glycan structures include particular modificationsthat are associated with truncation of the chains. These modificationsmay be due to bacterial activity present in the GLF sample as it isknown that bacteria can digest and consume glycans from proteins.However, such modifications can also be associated with disease,especially cancer where aberrant glycosylation has been linked to thedisease.

The foregoing analysis demonstrates that a large number of glycans canbe detected in samples obtained using a GLF.

Example 10—Detection of Metabolites in Samples Obtained Using a GLF

In this analysis, the ability of metabolites to be detected in samplesobtained using a GLF was assessed. To identify and analyze targetmolecules, including metabolites, in a GLF, a magnesium citrate-basedlavage composition was administered to human subjects, and the resultantGLF was analyzed for metabolites such as cholic acid and other bilesalts. The resultant GLF was collected from the subjects as part of acolonoscopy procedure.

3 ml GLF was centrifuged at max speed for 20 minutes and the supernatantacidified with 0.1% TFA. The supernatant was applied to a C18 SPE column(Waters Sep-Pak), washed with 3 volumes of 0.1% TFA, and eluted with 50%ACN in 0.1% TFA. The elutant was dried by centrifugal lyophilization andre-dissolved in 500 μl 0.1% TFA.

Data were acquired on a Waters Q-TOF mass spectrometer using input froman LC system. The A solvent contained 3% of B and 0.2% formic acid inwater. The B solvent contained 3% of A and 0.2% formic acid inacetonitrile. Solvents were HPLC grade from Fisher. The starting solventwas 5% B and remained for 5 min and then changed to 40% by 25 min, 90%by 30 min, and then reset to5% at 36. The MS scanned over the mass rangefrom m/z 100 m/z to 2000 every second. Data were acquired using thestandard MassLynx software. The eluting compounds with the cholic acidpeak marked are summarized in FIG. 2. A similar profile of peaks wasobserved on the Orbitrap instrument where the cholic acid peak wasidentified using a standard and MS/MS data. Metabolites including cholicacid were identified.

The foregoing analysis demonstrates that metabolites can be detected insamples obtained using a GLF.

The term “comprising” as used herein is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps.

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification are to be understood as beingmodified in all instances by the term “about.” Accordingly, unlessindicated to the contrary, the numerical parameters set forth herein areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of anyclaims in any application claiming priority to the present application,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

The above description discloses several methods and materials of thepresent invention. This invention is susceptible to modifications in themethods and materials, as well as alterations in the fabrication methodsand equipment. Such modifications will become apparent to those skilledin the art from a consideration of this disclosure or practice of theinvention disclosed herein. Consequently, it is not intended that thisinvention be limited to the specific embodiments disclosed herein, butthat it cover all modifications and alternatives coming within the truescope and spirit of the invention.

REFERENCES

Each of the following references is incorporated by reference herein inits entirety.

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All references cited herein, including but not limited to published andunpublished applications, patents, and literature references, areincorporated herein by reference in their entirety and are hereby made apart of this specification. To the extent publications and patents orpatent applications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

1. A method for assessing the physiological state of a subjectcomprising: obtaining a gastrointestinal lavage fluid and/or a fecalsample from the subject; and detecting a target molecule in thegastrointestinal lavage fluid and/or the fecal sample.
 2. (canceled) 3.The method of claim 1, wherein the gastrointestinal lavage fluid isobtained from the subject by partially purging the subject'sgastrointestinal system. 4-5. (canceled)
 6. The method of any one ofclaims 14, wherein the target molecule a) comprises a polypeptide,antibody, bile acid, metabolite, or glycan; b) comprises a biomarker,optionally, wherein the biomarker is associated with a disease, apositive response to treatment, a partial response to treatment, anegative response to treatment, or no response to treatment; c) isassociated with the presence of a cancer or predisposition to a cancer,optionally, wherein the cancer is pancreatic cancer, colorectal cancer,liver cancer, or gastric cancer; and/or d) originated from an accessorydigestive gland, optionally, wherein the accessory digestive gland is asalivary gland, pancreas, gallbladder, or liver. 7-12. (canceled) 13.The method of any one of claim 1, further comprising administering alavage fluid to the subject, optionally, a) wherein the lavage fluid isadministered orally; b) wherein the lavage fluid comprises an ingredientselected from the group consisting of polyethylene glycol, magnesiumsulfate, sodium sulfate, potassium sulfate, magnesium citrate, ascorbicacid, sodium picosulfate, and bisacodyl; and/or c) wherein the lavagefluid is selected from the group consisting of GOLYTELY, HALFLYTELY,NULYTELY, SUPREP, FLEET'S PHOSPHO-SODA, magnesium citrate, and theirgeneric equivalents. 14-16. (canceled)
 17. The method of claim 1,further comprising performing a colonoscopy on the subject.
 18. Themethod of claim 1, wherein the subject is mammalian optionally, human.19. (canceled)
 20. A method for identifying a biomarker comprising:obtaining a test gastrointestinal lavage fluid and/or a test fecalsample from a plurality of test subjects having a condition orphysiological state of interest and a control gastrointestinal lavagefluid and/or a control fecal sample from a plurality of control subjectswho do not have said condition or physiological state; determining thelevel of at least 5 target molecules in the test gastrointestinal lavagefluid and/or the test fecal sample and the control gastrointestinallavage fluid and/or the control fecal sample, and identifying a targetmolecule which is present at significantly different levels in the testgastrointestinal lavage fluid and/or the test fecal sample relative tothe levels in the control gastrointestinal lavage fluid and/or thecontrol fecal sample, thereby identifying a biomarker.
 21. (canceled)22. The method of claim 20, wherein the target molecules a) are selectedform the group consisting of polypeptides, bile acids, antibodies,metabolites, glycans, and a combination thereof; b) comprise abiomarker, optionally, wherein the biomarker is associated with adisease, a positive response to treatment, a partial response totreatment, a negative response to treatment, or no response totreatment; c) is associated with presence of a cancer or predispositionto a cancer, optionally, wherein the cancer is pancreatic cancer,colorectal cancer, liver cancer, or gastric cancer; and/or d) originatedfrom an accessory digestive gland, optionally, wherein the accessorydigestive gland is a salivary gland, pancreas, gallbladder, or liver.23. The method of claim 20, comprising determining the level of at least10, 20, 30, 50 or 100 target molecules in the test gastrointestinallavage fluid and/or test fecal sample and the control gastrointestinallavage fluid and/or the control fecal sample. 24-32. (canceled)
 33. Themethod of claim 20, further comprising administering a lavage fluid tothe test subjects and the control subjects optionally, a) wherein thelavage fluid is administered orally; b) wherein the lavage fluidcomprises an ingredient selected from the group consisting ofpolyethylene glycol, magnesium sulfate, sodium sulfate, potassiumsulfate, magnesium citrate, ascorbic acid, sodium picosulfate, andbisacodyl; and/or c) wherein the lavage fluid is selected from the groupconsisting of GOLYTELY, HALFLYTELY, NULYTELY, SUPREP, FLEET'SPHOSPHO-SODA, magnesium citrate, and their generic equivalents. 34-36.(canceled)
 37. The method of claim 20, further comprising performing acolonoscopy on the test subjects and control subjects.
 38. The method ofclaim 20, wherein the test subjects and control subjects are mammalian,optionally, human. 39-54. (canceled)
 55. A kit for detecting a targetmolecule in a gastrointestinal lavage fluid and/or a fecal samplecomprising: a lavage fluid for oral administration to a subject; avessel for collecting the gastrointestinal lavage fluid and/or the fecalsample from the subject; and an agent for detecting a target moleculewhich originated from outside the gastrointestinal system. 56.(canceled)
 57. The kit of claim 55, further comprising a proteaseinhibitor.
 58. The kit of claim 55, wherein the target molecule a)comprises a polypeptide, antibody, bile acid, metabolite, or glycan; b)comprises a biomarker, optionally, wherein the biomarker is associatedwith a disease, a positive response to treatment, a partial response totreatment, a negative response to treatment, or no response totreatment; c) is associated with presence of a cancer or predispositionto a cancer, optionally, wherein the cancer is pancreatic cancer,colorectal cancer, liver cancer, or gastric cancer; and/or d) originatedfrom an accessory digestive gland, optionally, wherein the accessorydigestive gland is a salivary gland, pancreas, gallbladder, or liver.59-64. (canceled)
 65. The kit of claim 55, wherein the lavage fluid a)comprises an ingredient selected from the group consisting ofpolyethylene glycol, magnesium sulfate, sodium sulfate, potassiumsulfate, magnesium citrate, ascorbic acid, sodium picosulfate, andbisacodyh; and/or b) is selected from the group consisting of GOLYTELY,HALFLYTELY, NULYTELY, SUPREP, FLEET'S PHOSPHO-SODA, magnesium citrate,and their generic equivalents.
 66. (canceled)